This application claims priority of Danish application 0188/96 filed Feb. 21, 1996, the contents of which is fully incorporated herein by reference.
The present invention relates to novel derivatives of naturally occurring insulins and analogues thereof which derivatives are soluble and have a protracted profile of action, to methods of providing such derivatives, to pharmaceutical compositions containing them and to the use of such derivatives in the treatment of diabetes.
Many diabetic patients are treated with multiple daily insulin injections in a regimen comprising one or two daily injections of a protracted insulin to cover the basal requirement supplemented by bolus injections of a rapid acting insulin to cover the requirement related to meals.
Protracted insulin compositions are well known in the art. Thus, one main type of protracted insulin compositions comprises injectable aqueous suspensions of insulin crystals or amorphous insulin. In these compositions, the insulin compounds utilized typically are protamine insulin, zinc insulin or protamine zinc insulin.
Certain drawbacks are associated with the use of insulin suspensions. Thus, in order to secure an accurate dosing, the insulin particles must be suspended homogeneously by gentle shaking before a defined volume of the suspension is withdrawn from a vial or expelled from a cartridge. Also, for the storage of insulin suspensions, the temperature must be kept within more narrow limits than for insulin solutions in order to avoid lump formation or coagulation.
While it was earlier believed that protamines were non-immunogenic, it has now turned out that protamines can be immunogenic in man and that their use for medical purposes may lead to formation of antibodies (Samuel et al., Studies on the immunogenicity of protamines in humans and experimental animals by means of a micro-complement fixation test, Clin. Exp. Immunol. 33, pp. 252-260 (1978)).
Also, evidence has been found that the protamine-insulin complex is itself immunogenic (Kurtz et al., Circulating IgG antibody to protamine in patients treated with protamine-insulins. Diabetologica 25, pp. 322-324 (1983)). Therefore, with some patients the use of protracted insulin compositions containing protamines must be avoided.
Another type of protracted insulin compositions are solutions having a pH value below physiological pH from which the insulin will precipitate because of the rise in the pH value when the solution is injected. A drawback is that the solid particles of the insulin act as a local irritant causing inflammation of the tissue at the site of injection.
WO 91/12817 (Novo Nordisk A/S) discloses protracted, soluble insulin compositions comprising insulin complexes of cobalt (III). The protraction of these complexes is only intermediate and the bioavailability is reduced.
Human insulin has three primary amino groups: the N-terminal group of the A-chain and of the B-chain and the xcex5-amino group of LysB29. Several insulin derivatives which are substituted in one or more of these groups are known in the prior art. Thus, U.S. Pat. No. 3,528,960 (Eli Lilly) relates to N-carboxyaroyl insulins in which one, two or three primary amino groups of the insulin molecule has a carboxyaroyl group. No specifically Nxcex5B29-substituted insulins are disclosed.
According to GB Patent No. 1,492,997 (Nat. Res. Dev. Corp.), it has been found that insulin with a carbamyl substitution at Nxcex5B29 has an improved profile of hypoglycaemic effect.
JP laid-open patent application No. 1-254699 (Kodama Co., Ltd.) discloses insulin wherein an alkanoyl group is bound to the amino group of PheB1 or to the xcex5-amino group of LysB29 or to both of these. The stated purpose of the derivatisation is to obtain a pharmacologically acceptable, stable insulin preparation.
Insulin analogues, which in the B30 position have an amino acid having at least five carbon atoms which cannot necessarily be coded for by a triplet of nucleotides, are described in JP laid-open patent application No. 57-067548 (Shionogi). The insulin analogues are claimed to be useful in the treatment of diabetes mellitus, particularly in patients who are insulin resistant due to generation of bovine or swine insulin antibodies.
U.S. Pat. No. 5,359,030 (Ekwuribe, Protein Delivery, Inc.) describes conjugation-stabilized polypeptide compositions for oral or parenteral administration comprising a polypeptide covalently coupled with a polymer including a linear polyalkylene moiety and a lipophilic moiety, said moieties being arranged so relative to each other that the polypeptide has an enhanced in vivo resistance to enzymatic degradation.
EP 511600 A2 relates i.a. to protein derivatives of the formula [protein] [Z]n wherein [protein] represents a protein having n amino residues each derivable from an amino group by removal of one of its hydrogen atoms, instead of amino groups, [Z] is a residue represented by the formula xe2x80x94COxe2x80x94Wxe2x80x94COOH wherein W is a divalent long chain hydrocarbon group which may also contain certain hetero atoms and n represents an average of the number of amide bonds between [Z] and [protein]. It is mentioned that the protein derivatives of the invention have an extremely prolonged serum half-life as compared with the proteins from which they are derived and that they exhibit no antigenicity. It is also mentioned, that insulin is one of the proteins from which derivatives according to the invention can be made, but no specific insulin derivatives are disclosed in EP 511600 nor is there any indication of a preferred [Z] or (a) preferred position(s) in which [Z] should be introduced in order to obtain useful insulin derivatives.
WO 95/07931 (Novo Nordisk A/S) discloses insulin derivatives in which the amino acid at position B30 is (a) a non-codable lipophilic amino acid having from 10 to 24 carbon atoms in which case the xcex5-amino group of Lysb29 has a lower acyl substituent or (b) any codable amino acid, in which case the xcex5-amino group of LysB29 has a lipophilic substituent or (c) deleted, in which case the xcex5-amino group of LysB29 has a lipophilic substituent. The insulin derivatives are soluble at physiological pH values and have a protracted profile of action.
By xe2x80x9cinsulin derivativexe2x80x9d as used herein is meant a peptide having a molecular structure similar to that of human insulin including the disulphide bridges between CysA7 and CysB7 and between CysA20 and CysB19 and an internal disulphide bridge between CysA6 and CysA11, and which has insulin activity. When the amino acid at position B1 is deleted, the position of the remaining amino acids of the B-chain are not renumbered.
Despite the many improvements already made in the field there still is a need for novel protracted injectable insulin compositions which are solutions and contain insulins which stay in solution after injection and possess minimal inflammatory and immunogenic properties.
One object of the present invention is to provide insulin derivatives soluble at physiological pH values and having a protracted profile of action.
Another object of the present invention is to provide insulin derivatives which have a long disappearance half-life from the injection site after subcutaneous injection.
A further object of the present invention is to provide a pharmaceutical composition comprising the insulin derivatives according to the invention.
A still further object of the invention is to provide a non-immunogenic insulin derivative.
A still further object of the invention is to provide a method of making the insulin derivatives of the invention.
A still further object of the invention is to provide a method of treating diabetes.
Surprisingly, it has turned out that certain derivatives of naturally occurring insulins and insulin analogues wherein the amino group of the N-terminal amino acid of the B-chain and/or the xcex5-amino group of LysB29 has a lipophilic substituent of the formula xe2x80x94COxe2x80x94Wxe2x80x94COOH as defined below have a protracted profile of action and are soluble at physiological pH values.
Accordingly, in its broadest aspect, the present invention relates to an insulin derivative as shown in formula I: 
wherein
Xaa at positions A21 and B3 are, independently, any amino acid residue which can be coded for by the genetic code except Lys, Arg and Cys;
Xaa at position B1 is (a) Phe, which is optionally substituted in the amino group with a substituent of the formula xe2x80x94COxe2x80x94Wxe2x80x94COOH wherein W is a divalent long chain hydrocarbon group having from 12 to 22 carbon atoms; or (b) deleted, in which case the amino group of Val at position B2 is either free or has a substituent of the formula xe2x80x94COxe2x80x94Wxe2x80x94COOH as defined above;
Xaa at position B28 is (a) Pro, in which case Xaa at position B29 is Lys which optionally, in its xcex5-amino group, has a substituent of the formula xe2x80x94COxe2x80x94Wxe2x80x94COOH as defined above; (b) Ser, in which case Xaa at position B29 is Lys which optionally, in its xcex5-amino group, has a substituent of the formula xe2x80x94COxe2x80x94Wxe2x80x94COOH as defined above; or (c) Lys which optionally, in its xcex5-amino group, has a substituent of the formula xe2x80x94COxe2x80x94Wxe2x80x94COOH as defined above, in which case, whether the xcex5-amino group of the Lys has the optional substituent or not, Xaa at position B29 is Pro;
Xaa at position B30 is (a) Thr; (b) Ala; or (c) deleted; and any zinc complexes thereof, with the proviso that the insulin derivative of formula I has at least one lipophilic substituent of the formula xe2x80x94COxe2x80x94Wxe2x80x94COOH as defined above.
In another aspect, the invention relates to an insulin derivative of the general formula I above wherein Xaa at position A21, B1 and B3 are as defined above, while Xaa at position B28 is Asp, Xaa at position B29 is Lys which, in its xcex5-amino group, has a substituent of the formula xe2x80x94COxe2x80x94Wxe2x80x94COOH as defined above and Xaa at position B30 is Thr.
In another aspect, the invention relates to an insulin derivative of the general formula I above wherein Xaa at position A21, B1 and B3 are as defined above, while Xaa at position B28 is Pro, Xaa at position B29 is Thr and Xaa at position B30 is Lys which, in its xcex5-amino group, has a substituent of the formula xe2x80x94COxe2x80x94Wxe2x80x94COOH as defined above and.
In one preferred embodiment of the invention, the divalent long chain hydrocarbon group, W, is xe2x80x94(CH2)12xe2x80x94.
In another preferred embodiment of the invention, the divalent long chain hydrocarbon group, W, is xe2x80x94(CH2)13xe2x80x94.
In another preferred embodiment of the invention, the divalent long chain hydrocarbon group, W, is xe2x80x94(CH2)14xe2x80x94.
In another preferred embodiment of the invention, the divalent long chain hydrocarbon group, W, is xe2x80x94(CH2)15xe2x80x94.
In another preferred embodiment of the invention, the divalent long chain hydrocarbon group, W, is xe2x80x94(CH2)16xe2x80x94.
In another preferred embodiment of the invention, the divalent long chain hydrocarbon group, W, is xe2x80x94(CH2)17xe2x80x94.
In another preferred embodiment of the invention, the divalent long chain hydrocarbon group, W, is xe2x80x94(CH2)18xe2x80x94.
In another preferred embodiment of the invention, the divalent long chain hydrocarbon group, W, is xe2x80x94(CH2)19xe2x80x94.
In another preferred embodiment of the invention, the divalent long chain hydrocarbon group, W, is xe2x80x94(CH2)20xe2x80x94.
In another preferred embodiment of the invention, the divalent long chain hydrocarbon group, W, is xe2x80x94(CH2)21xe2x80x94.
In another preferred embodiment of the invention, the divalent long chain hydrocarbon group, W, is xe2x80x94(CH2)22xe2x80x94.
Further preferred features of the present invention will appear from the appended claims.
Examples of preferred insulin derivatives according to the present invention are the following:
Nxcex5B29-(COxe2x80x94(CH2)14xe2x80x94COOH) human insulin and any zinc complexes thereof;
Nxcex5B29-(COxe2x80x94(CH2)16xe2x80x94COOH) human insulin and any zinc complexes thereof;
Nxcex5B29-(COxe2x80x94(CH2)18xe2x80x94COOH) human insulin and any zinc complexes thereof;
Nxcex5B29-(COxe2x80x94(CH2)20xe2x80x94COOH) human insulin and any zinc complexes thereof;
Nxcex5B29-(COxe2x80x94(CH2)22xe2x80x94COOH) human insulin and any zinc complexes thereof;
Nxcex5B29-(COxe2x80x94(CH2)14xe2x80x94COOH) ASpB28-human insulin and any zinc complexes thereof;
Nxcex5B29-(Coxe2x80x94(CH2)16xe2x80x94COOH) ASpB28-human insulin and any zinc complexes thereof;
Nxcex5B29-(Coxe2x80x94(CH2)18xe2x80x94COOH) ASpB28-human insulin and any zinc complexes thereof;
Nxcex5B29-(COxe2x80x94(CH2)20xc3x97CooH) AspB28-human insulin and any zinc complexes thereof;
Nxcex5B29-(COxe2x80x94(CH2)22xe2x80x94COOH) AspB28-human insulin and any zinc complexes thereof;
Nxcex5B30-(COxe2x80x94(CH2)14xe2x80x94COOH) AspB28L -humaninsulin and any zinc complexes thereof;
Nxcex5B30-(Coxe2x80x94(CH2)16xe2x80x94COOH) ThrB29LysB30-human insulin and any zinc complexes thereof;
Nxcex5B30-(COxe2x80x94(CH2)18xe2x80x94COOH) ThrB29LysB30-human insulin and any zinc complexes thereof;
Nxcex5B30xe2x80x94(COxe2x80x94(CH2)28xe2x80x94COOH) ThrB29LysB30-human insulin and any zinc complexes thereof;
Nxcex5B30-(COxe2x80x94(CH2)22xe2x80x94COOH) ThrB29LysB30-human insulin and any zinc complexes thereof;
Nxcex5B28-(COxe2x80x94(CH2)14xe2x80x94COOH) LysB28ProB29-human insulin and any zinc complexes thereof;
Nxcex5B28-(COxe2x80x94(CH2)16xe2x80x94COOH) LySB28ProB29-human insulin and any zinc complexes thereof;
Nxcex5B28-(Coxe2x80x94(CH2)16xe2x80x94COOH) LySB28ProB29-human insulin and any zinc complexes thereof;
Nxcex5B28xe2x80x94(COxe2x80x94(CH2)28xe2x80x94COOH) LySB28ProB29-human insulin and any zinc complexes thereof;
Nxcex5B28-(COxe2x80x94(CH2)22xe2x80x94COOH) LysB28ProB29-human insulin and any zinc complexes thereof;
Nxcex5B28xe2x80x94(COxe2x80x94(CH2)22xe2x80x94COOH)LysB28ProB29-human insulin and any zinc complexes thereof;
Nxcex5B29-(COxe2x80x94(CH2)14xe2x80x94COOH) desB30 human insulin and any zinc complexes thereof;
Nxcex5B29-(COxe2x80x94(CH2)16xe2x80x94COOH) desB30 human insulin and any zinc complexes thereof;
Nxcex5B29xe2x80x94(COxe2x80x94(CH2)18xe2x80x94COOH) desB30 human insulin and any zinc complexes thereof.
Nxcex5B29-(COxe2x80x94(CH2)20xe2x80x94COOH) desB30 human insulin and any zinc complexes thereof; and
Nxcex5B29xe2x80x94(COxe2x80x94(CH2)22xe2x80x94COOH) desB30 human insulin and any zinc complexes thereof.
Terminology
The three letter codes for the amino acid residues used herein are those stated in J. Biol. Chem. 243, p. 3558 (1968).
The expression xe2x80x9ca codable amino acidxe2x80x9d is intended to indicate an amino acid which can be coded for by the genetic code, i.e. a triplet (xe2x80x9ccodonxe2x80x9d) of nucleotides.
Preparation of the Compounds of the Invention
The compounds of the invention can be prepared by methods known per se. Thus, the group xe2x80x94COxe2x80x94Wxe2x80x94COOH of formula I can be introduced into an insulin moiety via an activated ester or an activated amide, e.g. an azolide, of the diacid HOOC-W-COOH. The preparation of activated esters is described i.a. in EP 0 511 600 A2 (Kuraray Co., Ltd.) and in WO 95/07931 (Novo Nordisk A/S). The preparation of azolides is described i.a. in W. Foerst, ed. Neure Methoden Der Prxc3xa4parativen Organischen Chemie, Band V, p. 53-93 (Verlag Chemie, Weinheim (1967)).
The group xe2x80x94COxe2x80x94Wxe2x80x94COOH can be introduced into an insulin moiety in which the amino group of the N-terminal amino groups of the A-chain and the B-chain is protected. This is in analogy with the methods described in in WO 95/07931. In this case a deprotection step follows the introduction of the group xe2x80x94COxe2x80x94Wxe2x80x94COOH as illustrated in the appended examples 1 and 2.
Alternatively, by selecting suitable reaction conditions as described e.g. in EP 0 712 862 A2, it is possible to introduce the group xe2x80x94COxe2x80x94Wxe2x80x94COOH selectively into the xcex5-amino group of a Lys residue without resorting to protection of the N-terminal amino groups of the A-chain and the B-chain. This is illustrated in the appended examples 3 and 4.
Experimental Results Achieved with the Compounds of the Invention.
Certain experimental data on the compounds of the invention are given in Table 1.
Lipophilicity
The lipophilicity of the insulin derivatives relative to human insulin, kxe2x80x3rel, was measured on a LiChrosorb RP18 (5 xcexcm, 250xc3x974 mm) HPLC column by isocratic elution at 40xc2x0 C. using mixtures of A) 0.1 M sodium phosphate buffer, pH 7.3, containing 10% acetonitrile, and B) 50% acetonitrile in water as eluents. The elution was monitored by following the UV absorption of the eluate at 214 nm. Void time, t0, was found by injecting 0.1 mM sodium nitrate. Retention time for human insulin, thuman, was adjusted to at least 2t0 by varying the ratio between the A and B solutions. kxe2x80x2rel=(tderivativexe2x88x92t0)/(thumanxe2x88x92t0).
Determination of Disappearance Half-life, T50%, from the Injection Site after Subcutaneous Injection of an Insulin Derivative in Pigs.
T50% is the time when 50% of the A14 Tyr(125I)-labeled analogue has disappeared from the site of injection as measured with an external xcex3-counter (Ribel, U et al., The Pig as a Model for Subcutaneous Absorption in Man. In: M. Serrano-Rios and P. J. Lefebre (Eds): Diabetes 1985; Proceedings of the 12th Congress of the International Diabetes Federation, Madrid, Spain, 1985 (Excerpta Medica, Amsterdam, (1986) 891-96).
For use in the determination of T50% as described above, samples of the products to be studied were iodinated with 125I using the standard lactoperoxidase method and the TyrA14-labeled product was isolated by isocratic ethanol/tris HPLC.
Binding to Porcine Albumin.
The binding to porcine albumin was determined in an in vitro assay. The values given in Table 1 under the heading xe2x80x9cAlbumin bindingxe2x80x9d are relative to the reference compound EXA.
Pharmaceutical Compositions
Pharmaceutical compositions containing an insulin derivative according to the present invention may be administered parenterally to patients in need of such a treatment. Parenteral administration may be performed by subcutaneous, intramuscular or intravenous injection by means of a syringe, optionally a pen-like syringe. Alternatively, parenteral administration can be performed by means of an infusion pump. A further option is a composition which may be a powder or a liquid for the administration of the insulin derivative in the form of a nasal spray. As a still further option, it may also be possible to administer the insulin derivative transdermally.
Pharmaceutical compositions containing an insulin derivative of the present invention may be prepared by conventional techniques, e.g. as described in Remington""s Pharmaceutical Sciences, 1985.
Thus, the injectable compositions of the insulin derivatives of the invention can be prepared using the conventional techniques of the pharmaceutical industry which involves dissolving and mixing the ingredients as appropriate to give the desired end product.
Thus, according to one procedure, the insulin derivative is dissolved in an amount of water which is somewhat less than the final volume of the composition to be prepared. An isotonic agent, a preservative and a buffer is added as required and the pH value of the solution is adjustedxe2x80x94if necessaryxe2x80x94using an acid, e.g. hydrochloric acid, or a base, e.g. aqueous sodium hydroxide as needed. Finally, the volume of the solution is adjusted with water to give the desired concentration of the ingredients.
Examples of isotonic agents are sodium chloride, mannitol and glycerol.
Examples of preservatives are phenol, m-cresol, methyl p-hydroxybenzoate and benzyl alcohol.
Examples of suitable buffers are sodium acetate and sodium phosphate.
Preferred pharmaceutical compositions of the particular insulin derivatives of the present invention are solutions of hexameric complexes. Typically, the hexameric complexes are stabilised by two or more zinc ions and three or more molecules of a phenolic compound like phenol or meta. cresol or mixtures thereof per hexamer.
In a particular embodiment, a composition is provided which contains two different insulins, one having a protracted profile of action and one having a rapid onset of action, in the form of soluble hexameric complexes. Typically the hexameric complexes are stabilized by two or more zinc ions and three or more molecules of a phenolic compound like phenol or meta-cresol or mixtures thereof per hexamer. The complexes are mixtures of hexamers of the particular insulins and mixed hexamers in which the ratio between the two different insulins is from 1:5 to 5:1.
A composition for nasal administration of an insulin derivative may, for example, be prepared as described in European Patent No. 272097 (to Novo Nordisk A/S).
The insulin derivatives of this invention can be used in the treatment of diabetes. The particular insulin derivative to be used and the optimal dose level for any patient will depend on a variety of factors including the efficacy of the specific insulin derivative employed, the age, body weight, physical activity, and diet of the patient, on a possible combination with other drugs, and on the severity of the case. It is recommended that the dosage of the insulin derivative of this invention be determined for each individual patient by those skilled in the art in a similar way as for known insulins.
The present invention is further illustrated by the following examples which, however, are not to be construed as limiting the scope of protection. The features disclosed in the foregoing description and in the following examples may, both separately and in any combination thereof, be material for realizing the invention in diverse forms thereof.